1. Field of the Invention
The present invention relates to a method for assaying the concentration of antigen in a sample by a radioimmunoassay technique. More particularly, the present invention relates to an automated radioimmunoassay technique for measuring antigen concentration via a selective antibody-antigen reaction, under conditions which permit extremely rapid analysis.
2. Description of the Prior Art
Isotope displacement using specific antibodies to measure minute quantities of insulin was first reported by Yalow et al (Nature 184, 1648) which has led to the widespread practice of this technique for the analysis of a wide variety of biologically important substances. Radioimmunoassay has become a primary technique for the routine analysis of literally hundreds of biochemical and clinically important substances. Radioimmunoassay is now the method of choice for the analysis of many substances, because antibodies with very high selectivity and affinity can be produced which permit measurement of any desired compound in rather impure samples. The amount of impurity can, in many cases, be 10.sup.9 times that of the substance of interest and not interfere with the measurement. This extraordinary selectivity and ability to detect femtomole (10.sup..sup.-15 mole) quantities of substances has pushed the radioimmunoassay to the forefront of modern analytical chemistry.
If the radioimmunoassay has any limitations at all, it is the amount of manual labor and time required to obtain results. A typical assay first involves the combination of the unknown sample or standard, specific isotope tracer and antibody. This solution is then incubated in the cold or at room temperature for at least 20-30 minutes to as long as several days to obtain equilibrium between the antigen (ligand molecule being measured) and the antibody. The antibody bound ligand isotope is then separated from the solution. This is generally accomplished by addition of dextran coated charcoal to absorb the free ligand, by precipitation of the antibody-isotope complex with ammonium sulfate or ethanol or by some other technique such as molecular sieve chromatography. The isotope antibody complex is recovered after centrifugation or collection of a specific column fraction and the radioactivity determined usually in an automatic beta or gamma counter. The amount of unknown substance present is determined from standard curves constructed from standards measured at the same time. Increasing additions of unknown sample reduces the specific activity of the isotope tracer thus yielding less radioactivity bound to the antibody.
The manual processing of samples for radioimmunoassay is time consuming, costly and requires meticulous attention to detail. In one laboratory alone, 8000 to 10,000 test tubes per month may be used for radioimmunoassay purposes. The repetitive nature and high precision of these determinations is responsible for considerable variability in the quality and reproducibility of the results. It is obvious that the complete automation of this technique would, of course, be desirable. Several attempts to automate radioimmunoassays have only met with limited success.
For instance, in Johnson, U.S. Pat. No. 3,896,217, a method is provided wherein a sample containing an unknown concentration of a specific antigen, and containing a known concentration of the same antigen tagged with a radioactive isotope, is passed through a bed of an immobilized antibody which is specific in its reactivity for the antigen being detected. As the solution is passed through the bed, both tagged and untagged antigen are bound to the immobilized antibody. Since insufficient antibody is provided in the bed to react with all of the antigen in the solution, the solution passing through the antibody bed will contain both untagged and tagged antigen which is passed into a detector where the amount of unbound, tagged antigen present is measured. The bed is then washed with a regenerating solvent which extracts all of the bound antigen, and the released antigen is also passed into the detector for measurement of the quantity of tagged antigen which has been bound. The results are then correlated to a standard curve for determination of the concentration of untagged antigen in the original sample. This technique however, is not completely satisfactory since it requires a substantial time delay to effect extraction of the antigen which had been immobilized with the antibody, and consequently, is not completely amenable to rapid analysis, which would be necessary to effect analysis of a multiplicity of samples. In the Johnson technique, it is necessary to permit the antibody-antigen reaction to go to completion in order to get a viable static system. All of these requirements are time consuming operations.
Another approach to automated radioimmunoassay has been described by Ertingshausen et al (Clinical Chemistry 21, 1305, 1975). The technique involves the initial automatic pipetting of antibody and antigen reagents followed by precisely timed incubation of the mixed ingredients. Radioimmunoassays of samples are determined in a static system similar to other conventional techniques, and the concentration of antigen in the original sample is determined by the use of preobtained, standardized curves. This procedure is burdened with much the same manual operational procedure as other prior art techniques which do not permit the continuous and rapid determination of a multiplicity of antigen containing samples.
The conventional antigen measuring techniques have suffered from several disadvantages in that it has been difficult to determine in the measurement if a sufficient amount of antibody or isotopically tagged antigen is present to react with the expected quantity of antigen. Thus, valuable analysis time was often wasted by the necessity of waiting for results before it could be realized that insufficient antibody or tagged antigen is present for proper analysis. Moreover, the conventional procedures generally involve batch type analysis, which results in inefficient use of highly skilled labor and high costs of reagents.
A need therefore continues to exist for an automated radioimmunoassay technique for rapidly determining the concentration of a specific antigen in a sample by use of the antibody-antigen interaction.